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To demonstrate the therapeutic potential of low-intensity ultrasound, it is imperative to characterize the bio-physical interaction of living cells with alteration of ultrasound frequency which was an obscure topic thus far. Here, we used a microfluidic platform for in situ single cell analysis with bio-physical interaction to ultrasound frequency alteration in line with the fact that microfluidic channels to a large extent mimic the confinement effect induced by micro confinement of physiological pathways. In this article, with the help of series of single-cell direct observation, we show that low intensity ultrasound frequency alteration would reversibly perturb cell membrane structure and count for inherent cell oscillation. Together, these findings indicate that alteration of low intensity pulsed ultrasound (LIPUS) frequency, if applied to a microfluidic platform on the order of minutes, would produce a reversible effect on physical structures of living cells based on the system resonant frequency during and post exposure ultrasound pulsing.